The present invention relates to confocal microscopy for examination of objects, such as biological tissue, and particularly to a confocal microscope system for scanning below the surface of tissue, which utilizes heterodyne detection to produce confocal images of tissue sections. This invention is especially suitable for providing an instrument for dermal or surgical pathology applications.
Confocal microscopy involves scanning a tissue to produce microscopic images of a slice or section of tissue. Such microscopic imaged sections may be made in-vivo and can image at cellular resolutions. Examples of confocal scanning microscopes are found in Milind Rajadhyaksha et al., xe2x80x9cIn vivo Confocal Scanning Laser Microscopy of Human Skin: Melanin provides strong contrast,xe2x80x9d The Journal of Investigative Dermatology, Volume 104, No. 6, June 1995, pages 1-7, and more recently, in Milind Rajadhyaksha et al., xe2x80x9cConfocal laser microscope images tissue in vivo,xe2x80x9d Laser Focus World, February 1997, pages 119-127. These systems have confocal optics which direct light to tissue and image the returned reflected light. Such confocal microscope systems can focus and resolve a narrow width of tissue as an imaged section, such that tissue structures can be viewed at particular depths within the tissue, thereby avoiding evasive biopsy procedures for pathological examination of the tissue, or allow pathological examination of unprepared excised tissue.
Two parameters which effect the performance of confocal microscope systems in imaging tissue sections are the numerical aperture (NA) of the optics and the wavelength of the beam scanned through the tissue. The axial resolution, i.e., the thickness of the imaged section, and lateral resolution of confocal microscope systems are directly proportional to the wavelength of the light source and inversely proportional to NA2 (axial) and NA (lateral). In other words, the higher the NA, the thinner the imaged section, while the lower the NA, the thicker the imaged section. Both the axial resolution and the lateral resolution are optimized in a confocal microscope system suitable for pathological examination to the dimensions of the tissue structures, such as cells, which are of interest. As discussed in the Milind Rajadhyaksha et al. article appearing in Laser Focus World, February 1997, the use of a near-infrared light source between about 700 nm and 1200 nm and optics with a NA of about 0.7-0.9 have provided optimal results for imaging tissue sections with sufficient discrimination of cellular level structures. One problem with using optics providing NA values about this range is that they are large and expensive, particularly for the objective lens which focuses light into and collects light from the tissue, and are very sensitive to aberrations, such as introduced by the object being imaged. Accordingly, it is desirable to provide imaging of tissue sections in a confocal microscope using lower cost and smaller optics having a NA below 0.7 without sacrificing imaging performance, in particular depth discrimination and scattered light rejection.
Accordingly, it is a feature of the present invention to improve confocal microscopy by combining the depth response of confocal imaging with the coherence function of heterodyne detection using a synthesized beam of multiple wavelengths of light, such that lower NA confocal optics and inexpensive laser diode sources may be used. Heterodyne detection has been proposed for imaging in U.S. Pat. No. 5,459,570, which describes an apparatus using an optical coherence domain reflectometer for providing images of a tissue sample to perform optical measurements. However, this apparatus is limited in depth resolution and does not utilize confocal optics for microscopic imaging. Other optical systems have used multiple wavelengths of light, but are limited to generating interference patterns for visualizing fringes characterizing the surface of objects, such as shown in U.S. Pat. No. 5,452,088, which describes a multi-mode laser apparatus for eliminating background interference, and U.S. Pat. No. 4,632,554, which describes a multiple frequency laser interference microscope for viewing refractive index variations. Such interferometric-based optical systems have no confocal optics or heterodyne detection, nor do they provide imaging within a tissue sample. A confocal microscope using multiple wavelengths of light has been proposed in U.S. Pat. No. 4,965,441, but this microscope is limited to focusing at different altitudes for surface examination of an object and does not have heterodyne detection.
It is the principal object of the present invention to provide an improved confocal microscopy method and confocal microscope system for imaging sections of tissue using heterodyne detection.
It is another object of the present invention to provide an improved confocal microscope system for imaging tissue using a synthesized light source to produce a beam having different wavelengths, in which the synthesized light source combines beams from multiple light sources producing light at each of the different wavelengths.
It is another object of the present invention to provide an improved confocal microscope system for imaging which can use low NA confocal optics, such as below 0.7, while achieving imaging performance in terms of axial resolution equivalent to prior art confocal microscope systems using higher NA confocal optics, such as between 0.7 and 0.9.
Briefly described, the system embodying the present invention includes a synthesized light source for producing a single beam of light of multiple, different wavelengths from multiple laser sources, and a first beam splitter for separating the single beam into an imaging beam and a reference beam. The phase of the reference beam is modulated by an optical modulator, while confocal optics scan and focus the imaging beam below the surface of the tissue and collect returned light of the imaging beam from the tissue. A second beam splitter is provided for interacting the returned light of the imaging beam with the modulated reference beam to provide a combined return beam having heterodyne components. The heterodyne components in the return beam represent the spatial overlapping of the imaging and reference beams over the bandwidth of the different wavelengths produced by the synthesized light source. The return beam is received by a photodetector which converts the amplitude of the light of the return beam into electrical signals in accordance with such heterodyne components representative of the tissue section. The electrical signals are then processed by a controller, such as a computer, to produce an image of the tissue section on a display coupled to the controller.
To promote the interaction of the imaging and reference beams in the return beam, the path lengths of the imaging and reference beams are matched such that the difference between their path lengths are approximately equal to integer multiples of the separation of the peaks in the coherence function produced by the synthesized light source.
The performance of the system, in terms of the axial resolution of the imaged tissue section, depends on the numerical aperture (NA) of the confocal optics and the multiple, different wavelengths of the beam produced by the synthesized light source, such that lower NA optics can be used to provide an axial resolution previously afforded by confocal microscope systems using higher NA optics between 0.7 and 0.9.
The system improves confocal microscopy by combining the axial resolution of confocal detection and the axial ranging of heterodyne detection of light with a coherence function, that is preferably periodic, to provide an axial (depth) resolution that is an improvement over that provided by confocal or heterodyne detection alone. It is believed that the heterodyne components are produced by overlapping one of the peaks of the coherence function with the broader depth response of the confocal optics, while all the other peaks do not contribute to the image of the tissue due to suppression by the confocal depth response. The signal is spatially limited to a region of said tissue in the focal plane along which the confocal optics scan and focus the imaging beam in the tissue.